Persistence of Transgene Expression in Synovium

滑膜中转基因表达的持久性

• 批准号: 6800409
• 负责人: Steven C Ghivizzani
• 金额: $ 29.27万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-15 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6800409
• 关键词: Adenoviridae; Lentivirus; arthritis; biotechnology; cytokine receptors; fibroblasts; flow cytometry; gene delivery system; gene expression; gene therapy; genetically modified animals; green fluorescent proteins; histocompatibility gene; immunogenetics; knee; laboratory rat; recombinant virus; synovial membrane; transfection; tumor necrosis factor alpha

DESCRIPTION (provided by applicant): Progress toward the clinical application of gene therapy for arthritis has been slowed by the inability to sustain intraarticular transgene expression. The prevailing observation is that transgene expression persists for only two to three weeks. The current proposal is designed to test the following hypotheses: 1) The immunologic incompatibility of foreign (non-self) transgene products leads to this premature termination of transgene expression, 2) Specific cell types within the synovium are capable of supporting stable, persistent transgene expression, and 3) In normal, immunocompetent animals, expression of cDNAs encoding homologous (self) or immunologically compatible gene products will persist long-term within the synovium. To determine the relative benefits and limitations of various ex vivo and in vivo gene delivery systems, each will be evaluated for transgenic persistence in the knees of athymic, nude rats. Because transduced cells that express foreign proteins survive for their natural life-span in this animal, it will serve as a "pseudohomologous" system within which we can use diagnostic marker genes to simulate the delivery of transgenes encoding "self" proteins to normal joints. Using the cDNA for Green Fluorescent Protein to phenotypically tag genetically modified cells in the synovium, we will use flow cytometry to characterize the populations of transduced cells. By studying how they change with time, we will identify the specific cell populations within the synovial lining that may permit long-term expression. The true motivation behind the study is however to determine for how long exogenous transgenes may be expressed in normal and arthritic joints in immunocompetent animals. For this, two different gene systems will be studied in normal rats that will not activate immune clearance. The first will be a soluble form of the rat TNF receptor type II; the second will be a "gene" construct that is transcribed, but not translated, avoiding altogether the immune issues of foreign protein products. The following specific aims will be addressed: 1) To comparatively evaluate synovial fibroblasts, dermal fibroblasts and mesenchymal stem cells for their capacity to enable persistent transgenic expression, and their biodistribution when used as vehicles for ex vivo intra-articular gene delivery to the knees of athymic nude rats, 2) To comparatively evaluate recombinant lentivirus, AAV and high capacity adenovirus for their capacity to enable persistent transgenic expression and their biodistribution when used as vehicles for direct intraarticular gene delivery to the knees of athymic nude rats, 3) To characterize genetically modified cell populations in the synovium and to determine how they change with time in vivo, and 4) Within immunocompetent rats, evaluate persistence of expression using immunocompatible transgene systems in normal and arthritic knees following ex vivo and in vivo delivery.

描述（由申请人提供）： 关节炎基因治疗的临床应用进展由于不能维持关节内转基因表达而减缓。普遍的观察是转基因表达仅持续两到三周。本提案旨在检验以下假设：1）外源性免疫不相容性（非自身）转基因产物导致转基因表达的这种过早终止，2）滑膜内的特定细胞类型能够支持稳定、持续的转基因表达，和3）在正常的免疫活性动物中，编码同源（自身）或免疫相容基因产物的cDNA的表达将在滑膜内长期存在。为了确定各种离体和体内基因递送系统的相对益处和局限性，将评价每种系统在无胸腺裸大鼠膝关节中的转基因持久性。因为表达外源蛋白的转导细胞在这种动物中存活了它们的自然寿命，它将作为一个“假同源”系统，在该系统中，我们可以使用诊断标记基因来模拟编码“自身”蛋白的转基因向正常关节的递送。使用绿色荧光蛋白的cDNA表型标记滑膜中的遗传修饰细胞，我们将使用流式细胞术表征转导细胞的群体。通过研究它们如何随时间变化，我们将确定滑膜衬里内可能允许长期表达的特定细胞群。然而，这项研究背后的真正动机是确定外源性转基因在免疫活性动物的正常关节和关节炎关节中表达的时间。为此，将在不会激活免疫清除的正常大鼠中研究两种不同的基因系统。第一种是大鼠TNF受体II型的可溶形式;第二种是转录但不翻译的“基因”构建体，完全避免了外源蛋白产物的免疫问题。将处理以下具体目标：1）为了比较地评估滑膜成纤维细胞、真皮成纤维细胞和间充质干细胞能够持续转基因表达的能力，以及它们在用作离体关节内基因递送至无胸腺裸大鼠膝盖的载体时的生物分布，AAV和高容量腺病毒在用作将基因直接关节内递送至无胸腺裸大鼠膝关节的载体时能够实现持续的转基因表达和它们的生物分布，3）表征滑膜中遗传修饰的细胞群并确定它们如何在体内随时间变化，以及4）在免疫活性大鼠中，评价在离体和体内递送后使用免疫相容性转基因系统在正常和关节炎膝关节中的表达的持久性。